This study is engaged in the detection and characterization of transient oxygenated intermediates which occur in biological oxygenases, with the aim of learning how molecular oxygen is activated in biological systems. Our current studies will focus on two basic enzymatic systems. The first is that of nonheme iron dioxygenases frequently found in bacteria. These enzymes cleave aromatic rings between adjacent phenolic groups. We have previously detected several intermediates involved in one such enzyme, protocatechuate dioxygenase. We shall be a) carefully studying the proton flux, in order to establish at which stages of the reaction protons are release or taken up; b) investigating alternative substrates and their reaction with the enzyme, and c) using EPR and rapid reaction freeze-quench techniques to better establish the chemical nature of these intermediates. A second similar enzyme, catechol dioxygenase, will also be studied by similar approaches. The second type of system is flavoprotein hydroxylases. The major effort will be concerned with the liver microsomal N,S-monooxygenase flavoprotein. We have already characterized intermediates involved in the reaction mechanism. We will try and extend these studies using various substrates, many of which are toxicologically and pharmacologically important. In addition we shall try and replace the FAD coenzyme with FAD analogues, with the intent of systematically varying the reactivity of the enzyme.